SOLUBLE-RNA OF PHAGE PHI-29 OF BACILLUS-SUBTILIS MEDIATES DNA PACKAGING OF PHI-29 PROHEADS ASSEMBLED IN ESCHERICHIA-COLI

The structural genes of the prohead of phage φ29 of Bacillus subtilis and a small φ29 RNA (sRNA) were cloned and expressed in Escherichia coli individually or in combination to study the role of the sRNA in prohead assembly and the mechanism of prohead morphogenesis. The genes coding for the proteins of the scaffold (gp7), the capsid (gp8), the portal vertex (gpl0), and the dispensable head fiber (gp8.5) were expressed in E. coli and the gene products were assembled, with and without the presence of the sRNA, into uniform and prolate particles that resembled the typical native φ29 prohead. No differences in particle size and shape were found between the particles of 7-8-8.5-10 (scaffoldcapsid-fiber-portal vertex) and 7-8-8.5-10-RNA (scaffold-capsid-fiber-portal vertex-RNA), suggesting that the φ29 sRNA was not required for φ29 prohead assembly. The 7-8-8.5-10 particles produced in E. coli in the absence of φ29 sRNA were fully competent to package φ29 DNA in the defined in vitro DNA packaging system by the addition of purified sRNA. Moreover, these DNA-filled heads were assembled into infectious virions in extracts. Without the addition of the sRNA, the 7-8-8.5-10 particles were incompetent while the 7-8-8.5-10-RNA particles were competent in DNA packaging. Bacterial sRNA present in E. coli cannot ubstitute for the φ29 sRNA. The assembly of prohead particles in E. coli indicated that host factors unique to B. subtilis were not required. The evidence that the φ29 sRNA was not required for φ29 prohead assembly and was not a fixed structural component of the φ29 prohead favors the conclusion that the 029 sRNA is a specific enzyme or morphogenetic factor in DNA packaging. © 1991.